Human papillomavirus vectors for the episomal transduction of host cells and method of making same

ABSTRACT

A replicon for delivery of a transgene for episomal gene expression in a mammalian host cell includes a transgene having an open reading frame or other nucleic acid sequence for transcription into RNA and under the transcriptional control of a first surrogate promoter and a first gene sequence expressing a papillomavirus replication initiator protein E1 tinder tile control of a second surrogate promoter. The replicon also includes second gene sequence expressing the papillomavirus replication origin binding protein E2 under the control of a third surrogate promoter, wherein tile transgene, the first and the second sequences are incorporated within at least one plasmid and less than three plasmids.

GRANT REFERENCE

The subject invention was made with government support under a grantfrom the National Institutes of Health/National Cancer Institute, GrantNo. CA36200 and a grant from the Cystic Fibrosis Foundation ResearchDevelopment Program—Component 2, Grant No. R464. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to modified human papillomavirus vectorsand to methods of making and using the same, as non-integrating episomalplasmids and to certain DNA sequences, inserted therein or expressedwithin a host cell of the viral vector. In particular, the inventionrelates to a papillomavirus, in particular human papillomavirus, inwhich a naturally occurring genome of the virus has been altered (“HPVvector”); to methods of making a vector and transducing cells therewith;to assure persistence of a vector, as an autonomous, extrachromosomalreplicon maintained by the viral E1 and E2 replication proteins, despitethe removal of all viral immortalization and transformation genes; andto certain DNA sequences inserted into or expressed in a host cell byway of an HPV vector, for purposes of long term gene transduction andexpression for applications in genetic modification and therapy.

BACKGROUND OF THE INVENTION

Since the development of recombinant DNA technology some 25 years ago,the prospect of developing extra-chromosomal gene expression vectorscapable of long-term expression in transduced cells and/or tissues hasbecome a reality with this invention. Prior developments in gene therapyresearch utilized vectors which either integrated the vector genome intothe host chromosomes or were replication defective and non-persistent.The integration of the vector genome into a host chromosome is apotential and real cause of chromosomal insertional mutations and isvulnerable to unpredictable or uncontrollable inactivation of a reportergene or a replacement gene. Retroviral vectors and adenovirus associatedvirus (AAV) vectors are representative examples of such insertionalvectors. Alternatively, replication defective vectors may provide forcytoplasmic expression of vector genetic material within a host cell,but at the expense of the transduction behavior being lost over time inproliferating cells. Defective adenovirus and virus vectors areillustrative of vectors capable of transient expression in eukaryotichost cells, but with transduction being lost in proliferating cells.Other widely recognized deficiencies of certain viral vectors are theirimmunogenicity, and complications due to (a) prior immunity leading toimmediate rejection of the infected cells and (b) induction of acuteimmune or hyperimmune responses within the first or second exposuresduring delivery of the vector to a host organism.

Thus, a novel vector capable of episomal replication transgene andregulated transcription can facilitate persistence and expression of atransgene in host cells without the limitations associated withinsertional mutagenesis or disregulation or transgene expressiondisruption due to integration. These limitations have been observed inmany in vivo studies using adenovirus or AAV vectors. J. M. Wilson, NewEng. J. Med. 1996, 334: 185-187. The human papillomaviruses have severalattractive properties that are useful in augmenting gene expression andextrachromosomal maintenance in transfected cells.

The large family of papillomaviruses are significant human pathogensthat infect epithelial tissues at selected body sites. Upon primaryinfection, or subsequent immunosuppression, papillomaviruses typicallyinduce overt lesions variously called warts, papillomas or condylomata.These may go into short or long term remission. Nevertheless, viral DNAcharacteristically persists as episomes in stem cells. The episomalviral DNA may reactivate upon immunosuppression. A. Ferenczy et al., NewEng. J. Med. 1985; 313: 784-788, and A. Maran, Virology 1995; 212:285-294. The latent phase of HPV infections is readily detected byPolymerase Chain Reaction (PCR) amplification of DNA. High levels ofviral DNA amplification and mRNA transcription typically requireterminal differentiation of squamous epithelia, and encapsidation intodaughter viruses occurs in the superficial keratinocytes, prior tokeratinocytes sloughing off the surface of the lesion. L. T. Chow and T.R. Broker, Viral Pathogenesis, N. Nathanson (Ed.), Lippincott-Raven,1996, pp. 276-302. Well over 30 genotypes of human papillomavirus havean affinity for mucosal epithelia. E. M. de Villiers, Clinics inDermatology 1997 (G. Orth and S. Jablonska, eds); 15: 199-206. Among themucoscotrophic human papillomavirus strains are HPV-6, HPV-11, andHPV-16. While infections with HPV-16 occasionally progress to high-gradelesions and carcinomas, infections by HPV-6 or HPV-11 are almostuniversally benign and rarely reach such an advanced level ofprogression. In fact, most humans are infected with one or more types ofcutaneous or mucosal HPV during their lifetime, and the virus persiststhereafter under a degree of immunological surveillance and control, butwithout complete elimination. In almost all cases, such persistentmaintenance remains subclinical with a small possibility of overtsporadic viral expression. For this and other reasons, HPVs areattractive vectors for long term gene therapy.

The HPV genome is a closed circular double-stranded DNA molecule,typically 7.9 kilo base pairs (kb) long. All HPV genotypes have asimilar genome organization. The genomic organization of HPVs is knownto the art and a representative prototype thereof, HPV-11, is set forthin FIG. 1. HPV genomes replicate as multi-copy nuclear, extrachromosomalplasmids. The differences in pathogenesis among various strains ofpapillomavirus mainly reside in the viral encoded E6 and E7 genes and inthe upstream regulatory region (URR).

The lesions and possible carcinomas associated with papillomavirusinfections are not associated with expression of HPV E1 and E2replication proteins; rather the severe pathogenesis is initiated byinopportune expression of the viral oncogenes E6 and E7. The high riskoncogenic HPV-16, HPV-18 and related virus strains can causedisregulation in cell growth, cell differentiation and apoptosis. Thenatural functions of the oncoproteins are to reactivate the host DNAreplication machinery in order to facilitate viral DNA amplification indifferentiated keratinocytes that otherwise no longer express the hostreplication proteins. Consistent with these functions, HPV E6 and E7genes are normally under the control of a differentiation-dependentupstream regulatory region (URR) promoter.

The E7 protein interacts with and inactivates the retinoblastomasusceptibility protein, pRB, a host tumor suppressor protein. M.Scheffner, et al. Curr. Top. Microbiol Immunology, 1994; 186: 83-99.This and/or related interactions reactivate the entire host DNAreplication machinery in differentiated cells so that the virus DNA canamplify. Host DNA also replicates under these conditions as a by-productof the action of E7. S. Cheng, et al. Genes Dev. 1995; 9:2335-2349. TheE6 protein inactivates another tumor suppressor protein, p53, therebyallegedly inhibiting premature apoptosis mediated by p53 whenunscheduled DNA replication occurs in the differentiated cells inresponse to HPV E7 function. The cumulative result of HPV E6 and E7protein activity in the differentiated stem cells can, in combinationwith presently unknown mutations in cellular genes, result in theimmortalization of human keratinocytes in vitro or in host lesions.These proteins also are capable of transforming primary rodentepithelial cells in concert with an activated oncogene such as c-ras orc-fos. L. T. Chow and T. R. Broker, Ibid; and M Scheffner, et al., Ibid.

Downstream of the E2 gene in an HPV genome is the E5 gene, as shown inFIG. 1. HPV E5 protein is not an oncogene; however, it enhances thesignal transduction of the EGF (epidermal growth factor) receptor, andcan cause other membrane alterations. H. Stoppler, et al. Intervirology1994; 37:168-179. Thus, HPV E5 protein augments the immortalizationfunction of E6 and E7, yet E5 alone is not capable of immortalizingprimary cells. M. C. Stoppler, et al., Virology, 1996; 223:251-254.While the role of HPV E5 is not fully appreciated in the literature, thebovine papillomavirus type 1 (BPV-1) E5 is the major viral oncogene. Akey distinction from the HPVs, BPV-1 E5 functions to transformestablished rodent cell lines in vitro. BPV-1 E5 is detected in basal aswell as in suprabasal differentiated cells of bovine warts caused byBPV-1. In light of the pathogenic nature of BPV E5 protein as well asother BPV proteins, such vectors raise a concern about the potential ofE5-like proteins for oncogenesis. Previous attempts to design a viralvector suitable for episomal expression of foreign DNA in eukaryoteshave met with only limited success due partly to the inability toseparate deleterious viral genes from essential viral regulatorysequences. The instant invention largely overcomes these difficultiesconcerning vector design because the HPV oncoproteins are distinct fromthe HPV proteins necessary to support DNA replication from the nativeviral origin sequences. L. T. Chow and T. R. Broker, Ibid. The E6 and E7viral oncogenes are normally up-regulated upon differentiation in HPVinfected cells.

Similarly, HPV E4 protein function is not fully understood. E4 is knownto associate directly or indirectly with cytokeratin intermediatefilaments, but additional functions are postulated to exist. It isknown, however, that HPV E4 is not an oncogene.

In contrast, to other members of the papovavirus family, such as mousepolyomavirus, simian virus 40, and human BK and JC viruses, for whichthe viral replication initiator and the oncogenic proteins are one andthe same, HPV DNA replication utilizes host-encoded DNA replicationenzymes, illustratively including DNA polymerases α and δ, PCNA, RF-C,RPA, DNA ligases and topoisomerases I and II. In addition, HPVreplication utilizes host enzymes for the synthesis ofdeoxyribonucleoside triphosphate substrates. The virus contributes threecomponents that are highly conserved among the papillomaviridae, whichinclude: (1) the origin sequence (ori), (2) the ori recognition proteinE2; and (3) the initiator protein E1. L. T. Chow and T. R. Broker,Interviroloy 1994, 37: 150-158; and A. Stenlund, DNA Replication inEukaryotic Cells 1996: pp. 679-697.

The ori consists of binding sites (BS) for the two viral proteins E1 andE2 and is located within a noncoding, transcription regulatory region ofapproximately 750-1000 base pairs, designated the upstream (5′)regulatory region (URR). The URR also contains numerous hosttranscription factor binding sites that are thought to confer tissuetropism. H. U. Bernard and D. Apt, Arch. Dermatol. 1994, 130:210-215. E2protein binds tightly to the multiple E2BS in the URR and helps recruitE1 to the origin. In addition, upon binding to E2BS, E2 also functionsas a transcription factor and activates a surrogate promoter at adistance, depending on its concentration and those of other, hosttranscription factors. Hirochika et al., Genes Dev. 1988, 2:54-67;McBride et al., J. Biol. Chem. 1991, 225:18411-18414; Ham et al., Trendsin Biochemical Sciences 1991, 16:440444. In its capacity as areplication and transcription factor, E2 bound to E2BS preventsnucleosome formation around the origin and help recruit E1 and perhapshost proteins to the origin to establish an initiation complex. Acomplex of approximately 6 copies of E1 which dimerizes to form a doublehexamer is a helicase and helps unwind the ori and recruit the host DNApolymerase a to initiate replication. The E1 complex is requiredcontinuously during the elongation phase of replication, unlike thetransient requirement for E2 during assembly of the pre-initiationcomplex on the origin. Because of sequence and functional conservation,E1 and E2 proteins encoded by the same virus can promote the replicationof either a homologous or a heterologous papillomaviral origin. C. M.Chiang et al., Proc. Natl. Acad. Sci. USA 1992, 89:5799-5803; A. DelVecchio et al., J. Virol. 1992, 66:5949-5958; F. Sverdrup and S. A.Khan, J. Virol. 1994, 68:505-509. Furthermore, replication from an HPVorigin is not restricted to human or epithelial cells as long asadequate levels of E1 and E2 proteins are produced/generated fromexpression vectors. C. M. Chiang et al., Ibid. This is the basis fortransient replication in transfected culture cells. M. Ustav and A.Stenlund, EMBO, J. 1991, 10:449-457. From their biochemical properties,it is clear that the E1 and E2 viral replication proteins are notoncoproteins.

The current knowledge of the mechanisms of HPV pathogenesis and oforigin-specific DNA replication are utilized in the instant invention todesign HPV based vectors without the risk of introducing viral oncogenesinto a host cell. Thus, the instant invention exploits the property ofHPV that the viral replication components and viral oncogenes areseparable. The most commonly used strategies for delivering potentialtherapeutic genes into cells are based on retroviruses, adenoviruses,adenovirus-associated (parvo)virus (AAV), and recombinant DNA plasmids.Each has its limitations. Moreover, expression from the retroviral LTRtends to be down-regulated. The use of cellular gene promoters mayovercome the problem. For adenoviruses, the strong host immune reactionto the highly immunogenic capsid proteins may prevent repeatedapplication. AAV is limited by its size to approximately 5 kb ofinserted DNA, and it integrates into host chromosomes nonspecifically.Transfection of plasmid DNA into cells is relatively inefficientcompared to virus infection. Considerable efforts have been devoted toimprove the efficiency of DNA transfection. A. R. Thierry et al., Proc.Natl. Acad. Sci. USA 1996, 193:11454-11459; E. R. Lee et al., Human GeneTherapy 1996, 7:1701-1717; N. Oudrhiri et al., Proc. Natl. Acad. Sci.USA 1997, 94:1651-1656. But the effectiveness of plasmid DNA isadditionally restricted by the short duration in transfected cells forlack of replication of the transfected DNA. Papillomaviruses haveproperties that circumvent many of these impediments.

The instant invention is based upon the modification of the naturallyoccurring papillomavirus genome to produce vectors by rearrangement ofthe natural genome, by the removal of DNA from the genome, and by theintroduction into the naturally occurring HPV genome of foreign DNA.Foreign DNA is defined herein as DNA naturally occurring in an organismother than HPV, or of synthetic origin. The genetic information designedfor expression from the vector is introduced into a host eukaryote via atrophic vector. A vector of the instant invention represents aninnocuous eukaryotic cloning vector for the expression of foreign DNA.The proclivity of HPV to infect specific eukaryotic epithelial celltypes, the ability to separate the replication mediating viral genesequences from those sequences responsible for oncogenesis and cellulargrowth control, and the capability to regulate the expression of theviral replication genes and transgenes by using previously characterizedhost or viral enhancers and promoters form the basis of the instantinvention.

There exists a need for a gene therapy method that overcomes theproblems of low-level transgene expression and expression for only alimited duration. The present invention fulfills this long-standing needin the art.

SUMMARY OF THE INVENTION

Vectors and methods are provided for introducing genetic material intohost mammalian cells. More particularly, vectors and methods areprovided for transferring a transgene to mammalian epithelial cells byway of a vector derived from a papillomavirus, such that the transgeneundergoes episomal maintenance and expression. The instant inventionharnesses the selectivity of a papillomavirus for specific mammalianhost cell types, but is not restricted to epithelial cells. The episomalreplication characteristics of HPVs are utilized for episomal transgeneexpression. In preparing a vector of the instant invention, genesequences of the HPV genome which are oncogenic which otherwiseup-regulate cell growth and/or DNA replication are excised in the courseof preparing the vector.

A vector of the instant invention functions to deliver a transgene forepisomal gene expression in a host cell. A vector of the instantinvention generically contains: a transgene having an open reading frameor other desired genetic sequence and under the control of a surrogatepromoter, and gene sequences expressing the HPV or other papillomavirusviral replication initiator proteins E1 and E2, expression of theseproteins being controlled by another surrogate promoter, wherein thetransgene and viral replication protein gene sequences are eithercontained within a single plasmid or located on two separate plasmids,which are cotransfected as an operative vector. The expression of thetransgene, E1 and E2 all benefit by the presence of HPV URR. For thepurposes of modification to achieve transfection in diverse cell types,the expression of the E1 and E2 and transgenes is or can be regulated byother enhancer-promoter units.

Upon transfection, the vector of the instant invention persists as anautonomous replicon following cell division an/or differentiation. Thetransgene is selected to serve any number of utilities, illustrativelyincluding: correction of an autosomal or somatic genetic or metabolicdeficiency; expression of a viral or prokaryotic protein or immunogenicfragment thereof, expression of a polypeptide foreign to the host; andmodulation of: hyperproliferative diseases (such as cancers), apoptoticdiseases, and states of infectious diseases of viral, bacterial, orprotozoan origin for purposes of control, reversal, cure or vaccination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the prior art genomic organization of HPV-11, the most likelyfunctions of proteins and sequences are labeled herein, and the virus isable to encode more proteins than it has open reading frames due toalternative utilization of promoters, poly A sites and mRNA splicing.

FIG. 2 is a schematic illustrating a production method of a one plasmidreplicon of the instant invention and methods of using same to produceepisomally transduced mammalian host cells.

FIG. 3 is a schematic illustrating a production method of a two plasmidreplicon of the instant invention and methods of using same to produceepisomally transduced mammalian host cells.

FIG. 4 is a schematic illustrating the construction of p11LUC,pMT2-11URRE1 and pMT2-11URRE2, where p11LUC is derived from pGL3c byinserting the 1 kb HPV-11URR into the parental plasmid. pMT2-11URRE1 andpMT2-11URRE2 are expression vectors for E1 and E2 proteins derived frompMT2-11E1 and pMT2-11E2, respectively by inserting the 1 kb HPV-11URRinto the parental vector; AdMLP therein defines the adenovirus majorlate promoter.

FIG. 5 is a schematic illustrating the construction of representativesingle plasmid replicons containing the HPV-11URR as an origin, whereinall p16R series replicons express HPV-16 E1 and E2 proteins from thesame transcription unit. The p11-R1 replicon expresses epitope-tagged E1and the native E2 proteins from the same transcriptional unit, thetransgene herein is, for illustration, either the LacZ or the greenfluorescence reporter protein.

FIG. 6 is a transient replication assay of p11LUC in human 293 cellswherein p11LUC is electroporated into human 293 cells with pMT2-11E2alone, or with both pMT2-11E1 and pMT2-11E2. Low molecular weight DNA isharvested two days post-transfection and digested with the DNAreplication enzyme Stu I (−) to linearize p11LUC or with Stu I and Dpn I(+), where Dpn I degrades input DNA to yield clearly demonstrable unitlength newly replicated DNA. The arrow herein points to linearizedp11LUC.

FIG. 7 is a histogram illustrating luminescence augmentation of theluciferase expression by HPV-11 cis elements and trans factors in humanIB3-1 cystic fibrosis epithelial CF cell line. IB3-1 cells aretransfected with various plasmids (indicated by +) mediated byDOTAP:DOPE cationic liposomes. The activity from the parental clonepGL3c is used as a reference. Transfection with only E1 and E2expression vectors free of reporter genes serves as a negative control.Four days post-transfection, luciferase activities from triplicateexperiments are determined and plotted on a log scale. Bars indicatestandard error.

FIG. 8 is a transient replication assay of p11LUC in: (A) IB3- 1 cellsfour days post-transfection by plasmids and (B) in airways ofFVB/N-C57BL/6 mice three days post-transfection, wherein low molecularweight DNA is digested with Stu I alone (−), or with Stu I and Dpn I(+). The digestion products were then amplified by using the polymerasechain amplification reaction with primers targeted to the luciferasereporter gene. Only replicated plasmids can generate the 1.6 kb PCRproduct. The arrows in (A) and (B) point to the 1.6 kb product from PCRamplification.

FIG. 9 is a transient replication assay in 293 cells of single plasmidreplicons (panel A, lanes 1-6, panel B, lanes 3-6) or negative control(panel B, lanes 7, 8). The single plasmid replicon also supported thereplication of a second reporter plasmid which contained an HPV URR,demonstrating the two plasmid system (panel B, lines 1,2). The cellstransfected with plasmid (S) are as indicated, wherein two dayspost-transfection, assays are performed after digestion with the DNArestriction enzyme Hind III alone (−) or with Hind III (+) plus Dpn I(+). The probe for panel A is prepared by random priming of a pUCplasmid containing a portion of the HPV-11URR, whereas the probe usedfor panel B consisted of p16R-1 plus p11URR-LacZ; herein a dot signifiesHind III restriction fragments from replicated DNA.

FIG. 10 is a persistence assay of 1 plasmid replicons during passage intransfected 293 cells, after electroporation with plasmid (s), cells areplated at 30% confluence on 100-mm plates and are passaged by splittingon days two, four, six, eight (P1, P2, P3, P4). Low molecular weight DNAis harvested on day four, six, eight and ten, digested with Hind III,Southern-blotted and probed; herein a dot symbolizes Hind III fragmentsfrom plasmid DNA remaining in the cells.

FIG. 11 is an assay of β-galactosidase activity in 293 cells duringpassage; cells are transfected with 1-plasmid replicon p16R-1-LacZ (toppanels) or with negative control pCMV-LacZ (bottom panels) which doesnot replicate and then split 1:3 to 1:3.5 every two days over a periodof ten days, and activities are assayed on days two, four, six, eight,and ten (labeled P0, P1, P2, P3, P4).

DETAILED DESCRIPTION OF THE INVENTION

Vectors and methods are provided for introducing genetic material intotrophic host cells. More particularly, vectors and methods are providedfor transferring a transgene to mammalian epithelial cells by way of avector derived from a papillomavirus, such that the transgene undergoesepisomal maintenance and expression. FIG. 2 is a schematic drawingillustrating the methods of the instant invention, including theproduction of a single plasmid vector (replicon) and the use of a vectorof the instant invention to episomally transduce a mammalian host cell.The instant invention harnesses the selectivity of papillomavirus forspecific mammalian host cell types but is not restricted to humanepithelial cells. The episomal replication characteristics ofpapillomaviruses are utilized for episomal transgene expression. Inpreparing a vector of the instant invention, gene sequences of thepapillomavirus genome which are oncogenic are excised.

As referred to herein, the term “transgene” or “reporter gene” relatesto a nucleic acid, either naturally occurring or synthetic, whichencodes a sense or an antisense transcript; a ribozyme; triplex orquadriplex-forming RNA; an RNA complementary to functional RNAs,including: RNA components of enzymes, including telomerases andnucleases; or a protein product. The term “nucleic acid” is intended tomean natural and/or synthetic linear, circular and sequential arrays ofnucleotides, for example cDNA, genomic DNA (gDNA), mRNA,oligonucleotides, and derivatives thereof The phrase“operatively-linked” is intended to define attached in a manner whichallows for transgene transcription. The term “encoding” is intended tomean that the subject nucleic acid is capable of transcription andtranslation into either the desired polypeptide or the subject proteinor transcription into the desired RNA in an appropriate expressionsystem, such as when the subject nucleic acid is linked to appropriatecontrol sequences such as promoter and enhancer elements in a suitablevector and when the vector is introduced into an appropriate system orhost cell. The term “polypeptide” refers to an amino acid sequence whichcomprises a full length protein and fragments thereof The elements of areplicon are previously provided, and generally the term “replicon”defines herein a eukaryotic gene expression vector system that remainsextra-chromosomal upon introduction into an appropriate system or hostcell, the replicon containing a portion of the HPV genome capable ofpersisting episomally in a cell, transducing the host cell with atransgene and replicating, but lacking in oncogenic sequences.Conversely, the autonomous replicon is optionally designed to express an“anti-sense” transcript for the purpose of hybridizing to a naturallyproduced RNA to inactivate it or, through enzyme activity, to target ahost cell for destruction. Moreover, the HPV-based plasmid expressionsystem may be designed to produce an RNA product that can establish atriplex or quadruplex with DNA present in the transduced cell.

The process of producing a single plasmid vector of the instantinvention involves a series of steps. These steps include:

1. Incorporating the HPV URR fragment (origin and enhancer) andexpression cassette to express only HPV E1 and E2 proteins from theviral genomic DNA fragment into a basic vector that contains aselectable marker.

2. Incorporating a transgene into a separate expression cassette in thesame plasmid.

3. Transfecting the one-plasmid replicon into cell lines for testingplasmid persistence and transgene expression over time. Plasmidpersistence is established by digestions with certain one cut or few-cutrestriction enzymes plus Dpn I, using low molecular weight DNA recoveredfrom lysed cells, followed by Southern blotting or by PCR amplificationof the transgene.

4. Transfecting the one-plasmid replicon into immunocompromized SCIDmice or immune-competent mice for testing plasmid persistence andtransgene expression over time. Plasmid persistence will be establishedby digestions with certain one-cut or few-cut restriction enzyme and byDpn I, using low molecular weight DNA from lysed cells followed by PCRamplification of the transgene.

5. Depending on the outcome, the expression cassette for either E1 andE2, the transgene or both may be modified to modulate the expression todesired and efficacious levels.

FIG. 3, is a schematic illustrating a production method for a twoplasmid replicon of the instant invention. The production methoddetailed in FIG. 3 varies from that for a single plasmid replicon inthat:

the transgene is incorporated into a separate expression cassette in aplasmid other than the plasmid expressing E1 and E2 proteins; and thissecond plasmid contains the HPV replication origin and is therefore ableto replicate in response to E1 and E2 protein binding; and

the two plasmids expressing (E1/E2 or transgene) are cotransfected toeffect episomal transgene expression.

It is appreciated that the components in the one- and two-plasmidreplicons are completely modular, and different combinations are readilytailored to suit each specific application. The particular HPV URR isselected upon consideration of the target host cells. For the particularexample of the treatment of cystic fibrosis in airways, the HPV-11 URRis incorporated both as an origin for replicon replication and also asan enhancer for transgene expression in airway cells. For expression incells of the uterine cervix, tropism makes the HPV-16 URR or the HPV-18URR preferable. The HPV-16 URR and HPV-18 URR each contains four E2binding sites (E2BS) and one E1 binding site (E1BS) and each functionsas an origin of replication in the presence of any matched pair of HPVE1 and E2 proteins, illustratively including HPV-16 E1 and E2 proteins;HPV-18 E1 and E2 proteins; or even the bovine papillomavirus E1 and E2proteins. In some of the recombinant DNA constructions, a URR optionallyalso doubles as a promoter or optionally provides the polyadenylationsignals and polyadenylation sites. The multiple use of a URR in thedesign of a replicon makes it possible to construct replicons smallerthan otherwise would be possible. For these reasons, it is appreciatedthat the E1 and E2 protein pairs are derived from any of the human oranimal papillomaviruses. In the representative examples of the instantinvention in which both E1 and E2 proteins are expressed from the sameplasmid, the E1 is tagged with the EE-epitope only for the convenienceof cloning and assay. The URR plasmid is readily replicated using E1protein without the EE epitope tag.

A transgene expression cassette of the instant invention contains anenhancer-promoter, the coding region of one or more proteins (or otherdesired elements to be transcribed into RNA), and a 3′ polyadenylation(polyA) signal and polyA site. The enhancer-promoter for expressing theE1 and E2 proteins or the transgenes is varied so as to target aspecific host cell or tissue types to the desired or efficacious levels.

In particular, since E1 and E2 proteins are needed in dividing cells butnot in quiescent or differentiated cells, optionally an expressioncassette is selected which contains a promoter of a host replicationgene. For human hosts, these replication genes illustratively include:DNA polymerase α (Pearson et al., Mol. Cell. Biol. 1991, 11:2081-2095)or PCNA (Morris and Mathews, J. Biol. Chem. 1990, 265: 16116-16125). Inparticular, the DNA polymerase α gene is shut off in differentiatedcells (Moore and Wang, Cell Growth Diff. 1994, 5:485-494). Thus, theusage of a host replication promoter prevents the synthesis of E1 and E2proteins in non-proliferating cells and minimizes the host immuneresponses which could eliminate the transduced cells. The use of apromoter of host replication genes also ensures universality of itsexpression in proliferating human and animal cells regardless of celltypes, because the signals that control proliferation are highlyconserved in mammals and perhaps all the vertebrates.

The promoter for the transgene optionally also targets the desired cellor tissue types. Promoters of tissue- or cell-type specific genes (forinstance, epithelial cells, liver cells, brain cells, cells of theimmune system) are available, as are constitutive promoters forhousekeeping host genes (such as the β-actin promoter) or viralpromoters (such as the retroviral promoter from the Moloney MurineLeukemia virus, the Rous Sarcoma virus, the SV40 early promoter, thehuman cytomegalovirus immediate early promoter, and the adenovirus majorlate promoter, and the like), or differentiation-stage specific genes(such as high molecular weight keratins or filaggrin), or genescharacteristically turned on or upregulated in disease states. Most ofthese promoters have no species barrier. Conditional promoters are alsoavailable commercially, such as the “tet on” which is turned on inresponse to tetracycline.

The vectors of the instant invention are hereby termed “autonomousreplicons.” A replicon of the instant invention has the followingcharacteristics: (1) Expression of papillomavirus replication proteinsE1 and E2; (2) an HPV origin of replication (ori) in functional linkageto the HPV gene sequences encoding E1 and E2, this DNA/fragment alsocontains a promoter element, and optionally enhancer elements, (3) atransgene under the control of a separate constitutive or regulatablepromoter. It is appreciated that the papillomavirus genes and atransgene are located within a single plasmid, or alternatively areplicon of the instant invention involves multiple cooperatingexpression plasmids.

A schematic of a proof of principle three plasmid replicon system of theinstant invention is detailed in FIG. 4. It is appreciated that the 3 kbluciferase gene shown in FIG. 4 is an illustrative reporter gene and isreadily replaced with any transgene or segment thereof. It is understoodthat it is well known to the art how one proceeds in excising a giventransgene; cloning an alternative gene sequence DNA or other DNAelements illustratively including: anti-gene-—antisense, a sequence thatproduces RNA products, a sequence that serves as a binding site sink forinteractions with macromolecules or small molecules, and a sequence forthe purpose of modulating nucleic acid metabolism; and adapting a giventransgene for insertion into a plasmid of the instant invention usingrestriction enzymes. By way of illustration, a gene which codes for aprotein defective in humans stricken with cystic fibrosis, the CysticFibrosis Transmembrane Regulator (CFTR) gene represents such atransgene. Upon replicon introduction into a suitable host cell, thehost cell is transduced and expresses the transgene. For example, in ahuman suffering from cystic fibrosis (CF), the tropism of HPV forepithelial cells is exploited to transduce such cells of the respiratorytract so as to express the transduced CFTR gene which is deficient in CFsufferers. Other transgenes operative herein illustratively include:host cell genes that are otherwise lacking or defective, such as inautosomal genetic diseases; host cell genes that are otherwise lackingor defective that cause somatic deficiencies; enzyme encoding geneswhich are otherwise lacking or defective; structural protein genes whichare otherwise lacking or defective; viral genes or immunogenic fragmentsthereof, so as to impart immune response to the viral gene sequence ortranslation products of the sequence; host cell or foreign antigenencoding gene sequences; genes coding for immune system-mediatedclearance of malignant cells, such as histocompatibility antigens,cytokines or tumor growth factors; foreign genes for an enzyme capableof modifying a benign substrate to form a potent chemotherapeutic, suchas Pseudomonas exotoxin A, diphtheria toxin, ricin; genes capable ofprocessing prodrugs to drugs, such as herpes virus thymidine kinase orkinases that phosphorylate acyclic nucleoside phosphonates and E.colipurine nucleoside phosphorylase; and genes encoding for polypeptideshaving cell or systemic regulatory functions. Transgenes encoding forchromophoric or chromogenic products are also operative in the instantinvention and find cosmetic applications, such as for tattoos.

A transgene, or fragment of a transgene, operative in the instantinvention is produced and purified by any number of methods known to theart. A transgene is optionally produced synthetically or alternativelyby treating mRNA derived from the transcription of a transgene with areverse transcriptase so as to produce a cDNA version of the transgene,or a transgene is obtained by direct isolation from a gene bank, viralor plasmid transduction vector, or similar source.

A transgene of the instant invention is flanked by 5′ control elementsincluding an enhancer and a promoter, a translational control sequenceand 3′ elements illustratively including a polyadenylation signal andRNA stabilizing or destabilizing elements. In a three plasmid embodimentof the instant invention shown in FIG. 4, a luciferase reporter geneacts as a model transgene. The luciferase reporter is driven by a 5′flanking SV40 early promoter and enhancer. The reporter plasmid furtherincludes an origin of replication, derived from a bacterial vectorillustratively including the bacterial vector, pUC 19 and the entireHPV-11 URR, the URR containing several E2BS and one E1BS. A genesequence coding for ampicillin resistance is optionally included in thetransgene plasmid in order to amplify the plasmid during and after drugselection in E. coli. The other two plasmids making up a three plasmidreplicon system of the instant invention are expression vectors for HPVE1 and E2 proteins, each of which also contains the HPV URR (ori). TheHPV E1 and E2 proteins expressed are derived from the HPV-11 genome.Optionally, the E1 and E2 proteins expressed are derived from the HPV-16genome or from other prototypes. It is appreciated that HPV-6 isbiologically virtually indistinguishable from HPV-11 and illustrationsof the instant invention utilizing HPV-11 are essentially synonymouswith comparable constructions and strategies that are optionallyutilized with HPV-6 and numerous other somewhat more distantly relatedHPV genotypes. The expression vector plasmids pMT2-11E1 and pMT2-11E2support transient replication of the transgene/reporter gene containedwithin a separate plasmid or the same plasmid. Preferably, the 1 kbHPV-11 URR is inserted into the E1 and E2 expression plasmids, therebyyielding pMT2-11URRE11 and pMT2-11URRE2 of FIG. 4. The presence of theHPV-11 URR in the transgene plasmid increases luciferase expression byat least an order of magnitude as compared to pGL3c, the parentalplasmid in which the luciferase reporter is expressed from the SV40enhancer and early promoter. It is further preferred that the expressionof the HPV E1 and E2 replication proteins is controlled by a singlepromoter. In this way, the expression of E1 and E2 proteins iscoordinated and a preselected stoichiometry is maintained. Morepreferably, the transgene/reporter gene is controlled by an HPV URR, inaddition to other enhancers and promoters, such as those of SV40.

Several versions of 2-plasmid and single-plasmid replicon systems areshown in FIG. 5. In the 2-plasmid embodiment of the instant invention,one plasmid expresses E1 and E2 proteins and also contains an HPV URR(ori), whereas the second plasmid expresses a transgene and alsocontains HPV URR (ori). In cells cotransfected by both plasmids, whereinboth plasmids are replicated. In the one plasmid embodiment of theinstant invention, all elements are incorporated into the same plasmid.The single plasmid replicon is the preferred embodiment of the instantinvention owing in part to the efficiency of establishing a singleplasmid in a host cell, as compared to multiple, complementing plasmids.The plasmids shown in FIG. 5 range in size from 7.8 kb to 14.3 kb,although plasmids of greater than 25 kb are operative in the instantinvention. The features of the 2-plasmid and the single plasmidreplicons of the instant invention are shown in FIG. 5 as follows: (1)Each plasmid expresses both E1 and E2 proteins from a singletranscription unit, based on mapping data of HPV RNA isolated fromcondylomata. The E1 protein is thought to be translated from anunspliced message, whereas the E2 protein is translated from a splicedmessage which removes almost all the E1 coding region to about 100 ntsupstream of the E2 open reading frame. L. T. Chow et al., J. Virol.1987, 61:2581-2588, M. O. Rotenberg, Virology 1989, 172:489-497 and D.A. Palermo-Dilts et al., J. Virol. 1990, 65:3144-3149. In a singleplasmid replicon, transcription of E1 and E2 is under the control of thesame promoter, such as the adenovirus major late promoter, with theadenoviral tripartite untranslated RNA leader sequence at the 5′ endoperating to modulate translation efficiency. An adenovirus major latepromoter has previously been utilized in expressing E1 and E2 fromseparate plasmids. C. M. Chiang et al., Ibid. It is appreciated thatother tissue- and cell-cycle specific promoters are operative to driveE1 and E2 expression, illustratively including: Albumin promoter, PCNApromoter, and DNA pol α promoter. (2) One replicon expresses anepitope-tagged HPV-11 E1 protein known to function in transfected cellsand in a cell-free replication system (S. R. Kuo et al., J. Biol. Chem.1994, 269:24058-24065) and the native HPV-11 E2 proteins (p11R-1),whereas the remaining exemplary plasmids express the native HPV-16 E1and E2 proteins (p16R series). There are several reasons for choosingHPV-16 replication proteins to replicate the HPV-11 origin. For unknownreasons, in the transient replication assay, HPV-16 E1 and E2 proteinsreplicated HPV-11 ori-containing plasmids more efficiently than thehomologous HPV-11 E1 and E2 proteins. In carcinomas associated withHPVs, it is known that HPV-16 DNA is often found to exist asextrachromosomal plasmids, whereas the DNA of other virus types is oftenintegrated into the host chromosomes, teaching that the HPV-16 functionsmay confer useful degrees of efficiency or selectivity. (3) All plasmidsuse the entire HPV-11 URR as origin and regulatory region. HPV-11 has anatural tropism for airway cells, most probably conferred by theenhancer elements in the URR. H. -U. Bernard and D. Apt, Ibid.Persistence of the plasmid appears dependent on the copy number of E2BS.M. Pariisoo et al., Ibid. The 1 kb URR has 4 E2BS versus 1 or 2 copiesin a minimally operative origin of replication. Additional transcriptionfactor binding sites in the URR also may be important for equitableplasmid segregation during cell division. (4) Some of the repliconsshown in FIG. 5 also contain the bacterial lacZ gene or the greenfluorescence protein (EGFP) reporter gene under the control of acytomegalovirus (CMV) promoter. (5) To modulate the level ofreplication, the plasmids differ in either the presence or absence ofthe SV40 enhancer which affects expression of HPV replication genes andthe Adenoviral VA (viral associated) RNA genes that influence mRNAtranslation efficiency.

For a given ori plasmid, the level of plasmid replication is related tothe amounts of E1, provided a minimal amount of E2 is present. S-R Kuoet al., J. Biol. Chem. 1994, 269:24058-24065. The instant inventionutilizes viral protein expression modulation as inferred from the extentof plasmid replication to promote long term plasmid maintenance withhost cells.

Replicons having either HPV-11 or HPV-16 replication proteinsdemonstrate that individual expression vectors of HPV-16 E1 and E2proteins replicated an HPV-11 or HPV-16 ori-containing plasmid moreefficiently than did the wild type HPV-11 replication proteins expressedfrom the same expression vector. This difference has been attributed toa more efficient translation of mRNA with the 5′ untranslated region ofthe HPV-16 E1 message. N. Zou et al., J. Virol. 1998, 72:3426-3441.

A surprising result of the instant invention involves the importance ofreduced HPV E1 and E2 protein expression to promote long termmaintenance of extra chromosomal plasmids. A technique for reducing HPVE1 and E2 protein expression includes deletion or disablement of the VAgenes within a vector. Disablement of the VA genes inhibits excessivetranslation of mRNAs expressed from a promoter. The promoterillustratively including the adenovirus major late promoter (AMLP) asshown in p16R-2 of FIG. 5. The Ad VA RNA genes are known to influencetranslation efficiency from messages containing the 5′ tripartite leadersequences. R. J. Kaufman et al. Mol. Cell Biol. 1985, 5:1750. Thedeletion or disablement operates to moderate replicon copy number, suchthat numerous cell doublings occur and the plasmid is maintained basedon reporter gene activity. It is appreciated that other plasmids andpromoters incorporated therein also exhibit overly-efficient mRNAtranslation, and gene disablement to limit mRNA expression is operativein such instances.

Another technique for promoting long term maintenance ofextrachromosomal plasmids of the instant invention includes selection ofHPV-11 E1 and E2 genes for expression since HPV-11 genes are expressedless efficiently than from other genotypes such as HPV-16. For instance,as the P11R-1 replicon of FIG. 5 expresses HPV-11 E1 protein tagged witha glutamate rich (EE) epitope and the native HPV-11 E2 protein. ThisEE-E1 has a reduced replication activity relative to the wild type E1protein due to reduced protein level. Kuo et al., 1994. The persistenceof replicons of the instant invention in rapidly dividing cell linesover nonreplicating plasmids shows that replicons like these aremaintainable in transfected tissues in vivo, as cells do not divide asrapidly or the daughter cells become differentiated.

In one application, replicons of the instant invention deliver thetransgene, human Cystic Fibrosis Transmembrane Regulator (CFTR), tohuman airway cells for expression. Because the significant morbidity andmortality associated with cystic fibrosis occurs in the lungs of cysticfibrosis (CF) sufferers, (as well as in other secretory tissues) therehave been substantial efforts directed toward the development ofpulmonary CFTR gene replacement therapy in the disease. Six clinicaltrials using a non-viral approach have concluded that the CFTR gene canbe successfully transfected in human airways in vivo with cationicliposomes. E. J. Sorscher and M. J. Welch at the N. Amer. CysticFibrosis Mtg., CF Human Gene Therapy Sym. Orlando, Fla., 1996; E. J.Sorscher and M. J. Welch, Ibid; Caplen et al., Nature Medicine 1995,1:39-46; J. Logan et al., Gene Therapy 1995, 2:38-49; S. C. Hyde et al.,Ped. Pulm. Suppl. 1996, 13:264; D. R. Gill et al., Gene Therapy 1997,4:199-209; and D. J. Porteous et al., 29ed Pulm. Suppl. 1996, 13:266.Comparatively low potency liposomal vectors, such as DC-Chol, DMRIE, andDOTAP, have each transferred CFTR to the nasal airways. In specificcases, evidence for bioelectric correction with these prior art vectorshas been reported. These studies have shown that the nasal PotentialDifference (PD) abnormality in cystic fibrosis patients is improved by ahighly active plasmid containing strong promoters for CFTR expressionwhen it is delivered as naked DNA or in complexes with liposomes. Aliposomal formulation in such prior art studies was Lipid-67 (GenzymeCorporation, Framingham, Mass.), a reagent developed specifically forthe purpose of lower airway gene transfer of CFTR. E. R. Lee et al.,Human Gene Therapy 1996, 7:1701-1717. These initial results, followingone-time administration in nasal airways in CF patients, have led to thedevelopment of more advanced studies of re-administration to the cysticfibrosis nasal mucosa or one-time dosing in the lower airways. The firstlower airway administration study was performed at a dose of aerosolizedmaterial known to transfer reporter genes effectively to rodents andnon-human primates. This human study showed bioelectric correction ofthe lower airway potential difference (PD) abnormality in several humansubjects. E. Alton at the N. Amer. Cystic Fibrosis Mtg., CF Human GeneTherapy Sym. Orlando, Fla., 1996.

The levels of gene transfer required for bioelectric correction incystic fibrosis airways makes the instant invention the most promisingtreatment modality for this disease symptom.

In situ hybridization and antibody localization studies have indicatedthat target cells for gene replacement in cystic fibrosis airways shouldinclude submucosal gland cells and the epithelial cells lining the largeto medium sized airways. Indirect evidence from intestinal potentialdifference measurements in CF mice, from airway cells obtained viabronchoscopy of CF patients, and from model epithelial monolayerssuggest that low levels of CFTR (e.g., 1 mRNA molecule/epithelial cell;5-8% of cells in a monolayer; 5% of total CFTR in the murine intestine)are sufficient to overcome bioelectric defects in the disease. B. C.Trapnell et al., Proc. Natl. Acad. Sci. USA 1991, 88:6565-6569, J. F.Engelhardt et al., Nature Genetics 1992, 2:240-247; and J. R. Dorin etal., Gene Therapy 1996, 3:797-801. Pre-clinical studies in mice, rats,rabbits, pigs, and non-human primates and in vivo studies of CFTR genetransfer in humans with both viral and non-viral constructs have beenlimited by low levels of transgene expression. Although only a smallnumber of cells expressing a small amount of CFTR mRNA might besufficient for functional correction of CF chloride transport defects,(i.e. PD), other well described abnormalities in CF airways (forexample, the elevated potential difference attributable toamiloride-sensitive sodium transport) require a much larger percentageof corrected cells in order to normalize these particular surrogateendpoints. L. G. Johnson et al, Nature Genetics 1992, 2:21-25. Thedevelopment of means to further augment CFTR plasmid-based expression isan important issue based on the following considerations. 1) Not allpatients treated with non-viral plasmid-type prior art vectors havedemonstrated a bioelectric correction. 2) The level of improvementreported in prior studies among CF patients who exhibit improvementafter lipid-mediated gene transfer has not indicated complete orpermanent correction of chloride transport abnormalities. 3) Other CFdefects that may contribute to disease pathogenesis require higherlevels of CFTR transgene expression, either on a per-cell basis or froma standpoint of the percentage of cells corrected. L. G. Johnson et al,Ibid; and J. J. Smith et al., Cell 1996, 85:229-236. The instantinvention describes the use of human papillomavirus (HPV) basedreplicons to augment plasmid-based expression of transgenes (byapproximately two orders of magnitude or greater), in association withthe possibility of plasmid persistence in airway cells.

Upon formation of a replicon, delivery of the replicon to a host cell isrequired. It is well known to the art that naked DNA or DNA “packaged”in different formats such as with lipid micelles or in conjugates withadenovirus and/or polylysine are suitable for gene delivery into hostcells or animals for therapeutic development. There are several methodsknown to the art for delivery of the DNA contained within a replicon ofthe instant invention to host airway cells. Representative methods forreplicon delivery to host cells in vivo include: uncomplexed “naked”DNA, and DNA complexed within a lipid, and DNA nanospheres. Each ofthese particular delivery methods is more fully elaborated upon below.

In general, the theories and applications discussed above and shown inthe Examples below, are applicable to other papillomavirus typesincluding those specific for vertebrate animals such as cows, dogs,sheep, horses, etc. Due to the similarities in papillomaviruses acrosstheir host range.

In order to demonstrate more fully the advantages arising from thepresent invention, the following examples are set forth by way ofexample only and are not intended as limitations of the claimedinvention.

EXAMPLE 1 Up-regulation of Reporter Expression from a RepliconContaining the HPV-11 URR in a 3 Plasmid Replication System

Transfection of HPV-11 E1 and E2 expression vectors pMT2-11E1 andpMT2-11E2 supports transient replication of a third plasmid whichcontains the URR from any of the HPVs when cotransfected into epithelialor fibroblast cells from different species. C. M. Chiang et al., Ibid.Thus, the collaboration among E1, E2 and the host replication proteinsto initiate replication from a wide range of HPV origin sequences doesnot exhibit any species or tissue type specificity, providing thatadequate quantities of E1 and E2 proteins are expressed. The entireHPV-11 URR containing four E2BS and 1 E1 BS (or subsets orduplications/multiplications thereof) is incorporated into a plasmidcontaining a luciferase reporter driven by the SV40 early promoter,pGL3c (Promega) to generate p11-LUC, as shown in FIG. 4A. Two dayspost-infection with p11 LUC, pMT2-11E1 and pMT2-11E2 into human kidneyepithelial 293 cells, growing in vitro in a standard growth medium forthis cell line, low molecular weight DNA is harvested. The low molecularweight DNA is indicative of replicated plasmid DNA. This is revealed bya Southern blot with a luciferase probe after Dpn I and Stu I digestion.The reporter plasmids replicated transiently as indicated by the DpnI-resistant linear DNA after digestion with both Stu I and Dpn I. Theseresults are demonstrated in the lanes marked by “+” in FIG. 6. Stu Ilinearized the p-11LUC plasmid, (AGG/CCT, in the vector) while Dpn I,which recognizes a four-base pair sequence, (GA/TC) cuts transfected DNAinto small pieces at sites that are methylated when the DNA is isolatedfrom bacteria. However, plasmid DNA which has replicated in eukaryoticcells is unmethylated at the Dpn I sites and is therefore resistant toDpn I digestion. Human epithelial cell line 293 is cotransfected by thethree plasmid replicon of the instant invention by standardelectroporation techniques.

In contrast, when pMT-2-11E1 or pMT-211E2 plasmid is omitted, the linearDNA is only detected when the low molecular weight DNA is digested withStu I alone, as shown in the figures marked “−” in FIG. 6. In summary,FIG. 6 shows that Dpn I-resistant, linearized p-11LUC is only detectedwhen all three plasmids are cotransfected (left 2 lanes of FIG. 6), butnot when the E1 expression plasmid was omitted (right 2 lanes of FIG.6).

To further optimize this replicon system, the 1 kb HPV-11 URR isintroduced as a controlling element into the E1 and E2 expressionvectors, pMT2-11URRE1 and pMT2-11URRE2, respectively. FIG. 4B shows thestructure of these plasmids. When transfected into a CF cell line,IB3-1, as mediated by cationic lipids, (DOTA: DOPE), p11-LUC hadincreased reporter activity relative to the parental clone, indicatingthat the URR functioned as a transcription enhancer in the airway cellsas expected, based on HPV-11 tropism. The extent of the increasedactivity is shown in FIG. 7. Cotransfection with E2 further augmentedluciferase expression (FIG. 7), consistent with an interpretation thatthe URR also acted as an E2-responsive enhancer in the presence of E2protein. H. Hirochika et al., Ibid. When the reporter plasmid iscotransfected with the E1 expression plasmid, there is a comparableincrease in luciferase activity above that of the reporter plasmiditself (FIG. 7). When the reporter was cotransfected with both E1 and E2expression plasmids, an increase in reporter expression by nearly fourorders of magnitude was obtained in CF airway cells, indicating thatreplication took place in the transfected cells. Low levels of transgeneexpression is one of the features generally acknowledged to limitliposome-mediated gene transfer in vitro and in vivo. The HPV-basedreplicons of the instant invention significantly augment plasmidtranscription in CF airway cells and plasmid replication in vitro.

EXAMPLE 2 -Plasmid Replication in IB3-1 Cells

Southern blot hybridization detects extra-chromosomal replication onlyif a minimum of 15% of the cells are transfected. M. Ustav and A.Stenlund, Ibid. Over 40% of 293 cells are transfected byelectroporation, making the replication assay by using Dpn I/Southernblot hybridization feasible, as shown in FIG. 6. In contrast, less than5% of IB3-1 cells are transfected by lipid-mediated replicon transfer.This level of transfection is below the routine current limits ofdetection for the Dpn I-resistant, newly replicated DNA by Southern blotassay. In response, a PCR-based method is developed to investigateplasmid replication of a three plasmid replicon embodiment of theinstant invention in vitro and in vivo. PCR amplification (without Dpn Idigestion) has previously been used to demonstrate the presence oftransfected plasmid DNA in mouse tissues after injection withDNA-liposomes. A. R. Thierry et al., Proc. Natl. Acad Sci. USA 1995,92:9742-9746. IB3-1 cells are transfected with various combinations ofreporter plasmid, p11-LUC, pMT2-11URRE1 and pMT2-11URRE2, usingDOTAP:DOPE cationic liposomes. Low molecular weight DNA was harvestedfour days post-transfection and digested with either Stu I or with Stu Iand Dpn I. PCR amplification with primers targeted to a 1.6 kb region ofthe luciferase gene is performed as described in Example 7. When the DNAis not digested with Dpn I, as shown in the left panel of FIG. 4A, a 1.6kb PCR product is detected in all transfections, except when aluciferase reporter plasmid is omitted. When DNA is digested with Dpn I,a 1.6 kb band is only visible in cotransfection of p11-LUC and E1 and E2expression vectors, but not after transfection with pGL3c alone, orp11-LUC alone, nor with E1 and E2 expression vectors in the absence ofp11-LUC, as shown in the right panel of FIG. 4A. These results show thatp11-LUC replicated in at least a fraction of the transfected cells inthe presence of E1 and E2 expression vectors.

EXAMPLE 3 Three Plasmid Replication System in Mammalian Airways

Surface airway epithelial cells and alveolar epithelial cells are apredominant site of transfection with cationic lipids, but not allepithelial cells are transfected efficiently. Transfected surfaceepithelial cells represent only a fraction of the overall tissue in atotal lung extract. Because of the relatively low efficiency of liposomemediated transfection in a mammalian airway, such as that of a mouse, itis not possible to perform transient replication assays using the DpnI/Southern blot hybridization methods, as shown in FIG. 6.

In response, the modified polymerase chain reaction (PCR) based testjust described is used to test for plasmid replication in mouse airwaytissues. The low molecular weight DNA from total mouse airway tissue isextracted three days post-transfection with the plasmids, as describedin Example 2 in complex with GL-67 (Genzyme Corporation). After StuI/Dpn I digestions, PCR amplification generated a 1.6 kb fragment onlywhen all three plasmids of the replicon detailed in Example 2 arecotransfected, as shown in FIG. 8B, lanes 2. A 1.6 kb fragment is notgenerated when pGL3c is transfected alone, as shown in FIG. 8B, lane 1.This demonstrates successful transduction into some dividing cells andplasmid DNA replication in the same cells. A PCR based test notutilizing Dpn I digestion previously demonstrated persistence but notreplication of transfected DNA in mice, as reported by Thierry et al.,Ibid.

EXAMPLE 4 Three Plasmid Replicon Construction

The HPV-11 E1 and E2 expression vectors pMT2-1 E1 and pMT2-11E2 havebeen constructed previously. The construction and description ofpMT2-11E1 and pMT2-11E2 is provided in C. M. Chiang, et al. IbidpMT2-11URRE1 and pMT2-11URRE2 are constructed by inserting the 1 kbHPV-11 URR into the Stu I site in the SV40 enhancer. The 1 kb HPV-11 URRspans nucleotides 7072-7933/1-99 and is removed from clone 23-3 by HindIII digestion. The map of clone 23-3 is described in H. Hirochika, etal, ibid. 1988; 2:54-67; p11LUC is prepared by cloning the HPV-11URRfragment into pGEM-1 (Promega Corp.), excised by Bam HI-Nhe I doubledigestion, followed by insertion into the Bgl II/NheI sites in pGL3c(Promega Corporation), as per conventional techniques. pGL3c expressesthe luciferase reporter from the SV40 early promoter. The structures ofthese plasmids of the instant invention are shown in FIG. 4.

EXAMPLE 5 Gene Transfer into Cell Lines

0.5 μg of the reporter plasmid together with: 1) 5 μg each ofpMT2-11URRE1 and pMT2-11URRE2 or 2) 5 μg of pMT2-11URRE2 and 5 μg ofunrelated filler plasmid DNA (a control) are electroporated into 5×10⁶human 293 cells, as described in Chiang, Ibid. One-third of the cellsare plated onto a 100 mm Petri plate cultured in DMEM plus 10% fetalbovine serum (FBS) for 48 hr before harvesting the low molecular weightDNA. IB3-1 cells, airway cells derived from a CF patient (a gift of Dr.Pam Zeitlin, The Johns Hopkins University), are grown in 48 well platesin LHC-8 media (Biofluids, Inc.). The wells are transfected intriplicate with various combinations of pGL3c, p11LUC, pMT2-11URRE1,pMT2-11URRE2, and filler plasmid in a complex with DOTAP:DOPE cationicliposomes (Avanti) in a 4:1 lipid to DNA molar ratio as follows: a) 150ng of p11LUC and 600 ng each of E1 and E2 expression vectors, b) 150 ngof p11LUC and 1,200 ng of filler DNA, c) 150 ng of p11LUC with 600 ngeach of E1 or E2 expression vector and filler DNA, d) 150 ng of pGL3cand filler DNA, or e) 150 ng of filler DNA and 600 ng each of the E1 andE2 expression vectors.

EXAMPLE 6 Gene Transfer into Murine Cells

For in vivo gene transfer into FVB/N-C57BL/6 mice, lipid GL-67 (GenzymeCorp.) is complexed with the transgene DNA of interest. Preferably, themolar ratio of lipid to plasmid DNA for this purpose is 1:6. 108 μg ofGL-67 are mixed with 250 μg of p11LUC and 200 μg each of pMT2-11E1 andpMT2-11E2 in a carrier volume of 550 μl. In the control experiment, 250μg of pGL3 are mixed with 400 μg filler DNA. After a 15 minuteincubation at 30° C., 100 μl of the lipid:DNA mixture are pipetted onthe tip of the nares of lightly anesthetized mice over the duration ofone minute. Three animals are treated with each lipid:DNA formulation.Three days later, mice are sacrificed by CO₂ poisoning and their lungsand tracheas removed. The tissues are processed as described in A. R.Thierry, Ibid. Ten to thirty mg of the tissues are extracted for lowmolecular weight DNA for replication assays.

EXAMPLE 7 Plasmid PCR Replication Assays in Vitro

Transient replication assays in human 293 cells are conducted asdescribed in Chiang, et al. Ibid Human epithelial 293 cells are selectedfor replication and persistence because of the efficiency oftransfection in these cells. 2 days post-transfection, low molecularweight DNA is harvested by the alkaline Hirt lysis procedure. Half theDNA from a 100 mm plate is digested with Stu I which linearizes thereporter plasmid, while the other half is cut with both Stu I and Dpn I.The Dpn I/Southern blot is used to demonstrate replicationunequivocally. The digestion products are separated on a 0.8% agarosegel, Southern blotted onto a nylon membrane and then probed with theluciferase cDNA, which is ³²P-labeled by random priming. Anautoradiogram is developed after an overnight exposure.

PCR-based assays are used to detect plasmid replication in IB3-1 cellsand in mouse airway tissues. Low molecular weight DNA is harvested 4 and3 days post-transfection, respectively, and digested with restrictionenzyme or enzymes as just described. Thirty cycles of PCR amplificationare then conducted using primers5′-TAAAAAGCTTATGGAAGACGCCAAAAACATAAAGAAA and5′-GCCCAAGCTTATCGATTACACGGCGATCTTTCCGCCCTTCTT that target a 1.6 kbregion of the luciferase gene. Each cycle consists of 30 sec at 95° C.for denaturation, 30 sec at 60° C. for annealing, and 90 sec at 72° C.for polymerization. The products are then run in a 0.8% agarose gel andrevealed by ethidium bromide staining. A 1.6 kb PCR product amplifiedfrom the pGL3c plasmid is used as a positive control.

EXAMPLE 8 Luciferase Assay

Four days post-transfection of IB3-1 cells, cell lysates are harvestedwith 50 μl of luciferase assay buffer (Promega) and centrifuged toremove debris. The lysates are combined with luciferin reagent and readfor light (bioluminescent) activity in a luminometer with signalintegration for 10 sec., the results of which are indicated in FIG. 7.

EXAMPLE 9 One Plasmid and Two Plasmid Replicon Construction

The vectors that are used to construct the HPV single plasmid repliconsare pMT2 and pMTX. The HPV-16 E1 and E2 protein expression vector pMT2has been previously described. R. J. Kaufman et al., Molecular and CellBiol. 1989, 9:946-958; and Kuo et al., Ibid. The HPV-11 URR(7072-7933/1-99) is inserted into pMT2 at the Stu I site located withinthe SV40 enhancer, as shown in FIG. 5. This fragment of the HPV-11genome contains the poly-adenylation signal for late viral genes, andthe viral replication origin which overlaps the transcription regulatoryregion includes a promoter. Some representative single plasmid repliconsof the instant invention are shown in FIG. 5, the construction of whichfollows: To assemble p16R-1, the HPV-16 genomic DNA fragment fromnucleotides 686 to 4470 flanked by (Pvu I1 and Stu I) restriction sites,which includes the open reading frames (ORFs) of E1, E2, E4 and E5 aswell as the polyadenylation site for early genes, is cloned into thepMT2 vector at the Pst I and EcoR I sites. To prepare p16R-2, the VAgenes and the DHFR gene fragments are removed from p16R-1 by ligatingthe pMT2 fragment that spans the HPV-11 URR, SV40 ori/enhancer,adenovirus major late promoter, and HPV-16 genomic fragment containingbetween nucleotides 686 and 4470 into pUC19. To construct p16R-1-EGFP, athree-piece ligation was performed to piece together the Sal I-Spe Ivector fragment from p16R-1; the blunted ended Sal I-Ase I fragmentcontaining the CVM promoter and EGFP sequences from plasmid pEGFP-Cl(Clontech), and the Stu I-Spe I fragment which contains the SV40ori/enhancer, Adeno ML promoter, and HPV-16 nucleotides 686-1462 (SpeHI) from p16R-1. In this construction the transcription of greenfluorescence protein reporter (EGFP) gene uses the HPV-11 latepolyadenylation site within the URR fragment. The EGFP gene is replacedby the LacZ gene to construct p16R-1-Lac Z. The plasmid p11R-1 containsthe contiguous HPV-11 E1 and E2 ORFs from the genomic DNA (nts832-3844), except that the amino terminus of E1 is tagged with theglutamic acid-rich (EE) epitope from the polyomavirus middle T antigen.T. Grussenmeyer et al. Proc. Natl. Acad. Sci. USA., 1985, 82:7952-7954.To prepare p11R-1, the HPV-11 genomic DNA fragment (between nts 1380 and3844) is amplified by the polymerase chain reaction (PCR), cut with SphIand is ligated to the 5′ end of the EE-E1 coding sequences onpMTX-11EE-E1 SphI site (nt 1399) in the E1 ORF and EcoRI in the multiplecloning site of pMTX. pMTX is a derivative of pMT2 in which the multiplecloning site from pUC 19 was inserted into pMT12. S. R. Kuo et al.,Ibid, 1994. p11URR-LacZ contains the bacterial neomycin-resistance geneexcised from pLJd by digestions with SalI and ClaI. S. Cheng et al.(1995). In this clone, the HPV-11 URR serves multiple roles as theeukaryotic replication origin, the promoter of the LacZ gene as well asthe polyadenylation site for the neomycin-resistance gene. Thepurification of all the plasmids is performed by two rounds of bandingin CsCl-ethidium bromide equilibrium density gradient centrifugation.

In reducing the number of plasmids necessary to form a self-sustainingreplicon set, the efficiency of plasmid persistence is increased,relative to a multiple-plasmid replicon set. Some of the representativeplasmids shown in FIG. 5 contain a reporter gene such as the bacterialLacZ gene, or EGFP, controlled by the CMV promoter. Some of therepresentative replicons shown in FIG. 5 lack a reporter/transgene andare also useful in optimizing the levels of E1 and E2 proteins, asreflected by replicon copy number in transfected cells that an immunecompetent host tolerates.

EXAMPLE 10 Transient Replicon Replication Assays

Human kidney epithelial cell line 293 and human cervical carcinoma cellline C33 A are maintained in DMEM plus 10% calf serum in 5% CO₂ at 37°C. Transfections are conducted by electroporation, as describedpreviously. C. M. Chiang et al., J. Virol. 1992, 66:5224-5231. Atdifferent times post-transfection, low molecular weight DNA is harvestedby the alkaline Hirt method and treated with RNase. M. Ustav and A.Stenlund, Ibid Half of the low molecular weight DNA from one 100-mmplate is digested with Hind III, which cuts each plasmid two or threetimes. The other half is digested with both Hind III and Dpn I, whichcleaves the input DNA that did not replicate into smaller fragments. Asindicated above, the newly replicated DNA is resistant to Dpn Idigestion when these sites become unmethylated upon replication. Thedigestion products are separated in 0.8% agarose gels byelectrophoresis. The gels are Southern blotted onto a nylon membrane.The DNA is detected by hybridization with ³²P-labeled probes asspecified for each experiment and exposed to X-ray films orPhosphorImager plates. The probes are labeled by the random primingmethod (Amersham).

Each of the six single- or two-plasmid replicons shown in FIG. 5 iscapable of self-replication. p16-R-1 has the highest replicationefficiency and produced the highest copy number of the replicons.p11-R-1 replicates to a more moderate extent, as shown in FIG. 4A.Generally, larger replicons with a reporter gene/transgene replicate tolower copy numbers than similar replicons, as shown in FIG. 8B.Replicons which lack either the SV40 enhancer or the VA genes replicateto a much lower copy number, as compared to those replicons containingsuch sequences (not shown). The low copy number associated withreplicons lacking either the SV40 enhancer or the VA gene isattributable to the reduced transcription or translation of the E1 andE2 RNAs.

EXAMPLE 11 Persistence of HPV-16 R-1 and Reporter Gene Expression inTransfected Cells

The 14.3 kb p16R-1-lacZ and 11.2 kb p16R-1-EGFR 1-plasmid repliconstested for persistence in the transfected and proliferating 293 cellsover a 10-day duration are shown in FIG. 10. The cells are split atnear-confluence approximately 1:3 to 1:3.5 every 2 days. Low molecularweight DNA is harvested from each passage, and Southern blotting isperformed after Hind III digestion. Hind III cuts the various plasmidsinto several pieces. FIG. 10 shows the non-replicating pCMV-LacZ controlplasmid is barely detectable after four days indicating that, in theabsence of replication, most of the input DNA has been degraded by thattime. In contrast, the p16-R-1 replicons are still detectable 10 daysafter being split 4 times in culture indicating plasmid persistenceafter approximately 8 to 10 cell divisions. In addition, with the helperplasmid present, LacZ-positive cells also persisted for 10 days whereas,in cultures transfected with pCMV-LacZ in the absence of the helper,only occasional LacZ-positive cells are detected on days 6, 8 and 10, asshown in FIG. 11.

p16-R-1 replicons persist through repeated cell divisions over theduration of the experiments, as shown in FIGS. 9 and 10.

EXAMPLE 12 Inhibition of E4 Protein Expression

The expression of E4 messenger RNA or protein may have a deleteriouseffect on the copy number tolerance of host cells. The replicons of theinstant invention are optionally modified to limit the expression of theE4 mRNA or protein. The E4 splice acceptor site of the p16R-3 repliconof FIG. 4 has been site-mutated without changing the coding of theoverlapping E2 open reading frame. The site mutation is carried out andscreened for by standard PCR-mediated site-directed mutagenesis.

Alternatively, E4 protein synthesis is optionally prevented byintroducing a premature stop codon into the E4 open reading frame bymeans of a conservative amino change in the overlapping E2 open readingframe. The amino acid change and subsequent screening for successfulcodon introduction utilizes conventional techniques known to the art,including PCR and restriction fragment swapping as just described. Tominimize the size of the residual peptides translated, plasmids areselected in which the termination codon is introduced near the 5′ aminoterminal coding domain of the E4 open reading frame. When a terminationcodon is introduced into the E4 open reading frame with a conservativeamino acid change in the overlapping E2 open reading frame in the hingeregion of the E2 protein, the resulting replicon no longer expresses theE4 protein. Mutations eliminating E4 expression are confirmed bytechniques including antibody detection of E4.

EXAMPLE 13 Alternative Promoter for E1 and E2 Genes

In the preceding examples, the E1 and E2 replication genes are under thecontrol of the adenovirus major late promoter and contain the adenovirustripartite leader in the 5′ untranslated leader sequence. Alternativepromoters include the HPV11 URR or, preferably, a promoter of a hostreplication gene, such as in the case of a human host being that for theDNA polymerase a (Pearson et al., Mol. Cell. Biol. 1991, 11:2081-2095)or PCNA (G. F. Morris and M. B. Mathews, J. Biol. Chem. 1990, 265:16116-16125) is utilized, since E1 and E2 proteins are only needed to bemade in dividing cells (where those host replication genes are normallyexpressed) but not in quiescent or differentiated cells. A hostreplication gene promotor is regulated during the cell cycle, forexample, a DNA polymerase α gene is shut off in differentiated cells (A.L. Moore and T. S. Wang, Cell Growth Diff. 1994, 5:485-494). Thus, theusage of one such host promoter prevents the synthesis of E1 and E2proteins in non-proliferating cells and minimizes host immune responseswhich might eliminate the transduced cells. The use of the promoter ofthe host replication genes also ensures universality of its expressionin proliferating human and animal cells regardless of cell types,because the signals that control proliferation are highly conserved, inparticular among mammals and generally among all vertebrates.

EXAMPLE 14 Removal of E5 Gene Sequences from HPV-16 Based Replicons

The E5 gene(s) is already removed from and lacking in the wholly HPV-11origin replicons of the instant invention and can be removable from theHPV-16 replicons as well. The E5 genes are removed from the replicons ofthe instant invention, including those shown in FIG. 5, by PCR-mediateddeletion from E4 negative mutant clones, as per conventional techniques.

EXAMPLE 15 An Alternative Promoter for a Reporter Gene/Transgene

In those instances where the reporter/transgene protein is responsiblefor eliciting an immune response in vivo, the expression of such areporter/transgene protein is optionally down-regulated by a weakerpromoter than that of SV40. An example of a weaker promoter is theHPV-11URR itself The HPV-11URR has low activity in undifferentiatedkeratinocytes and is up-regulated in differentiated epithelial cells ofa mammalian host. Although HPV is trophic for epithelial cells, theHPV-11URR-driven reporter gene is present at only about 10% the level inmouse cell lines, as compared to human cells.

A transgene (or antisense sequence) is optionally expressed from apromoter of choice depending on the cell types to which the transgene istargeted. Promoters of tissue- or cell type-specific genes (for instanceepithelial cells, liver cells, brain cells, cells of the immune system)are available, as are constitutive promoters for housekeeping hostgenes, such as the β-actin promoter or viral promoters illustrativelysuch as the retroviral promoter from a Moloney Murine leukemia virus, aRous Sarcoma virus, a SV40 early promoter, a cytomegalovirus immediateearly promoter, an adenovirus major late promoter, and the like. Most ofthese representative promoters have no species barrier although theirefficiency varies depending on the host. Conditional promoters are alsooperative herein and available commercially, such as the “tet on” whichis turned on in response to tetracycline. For instance a transgene maybe selected to be a suicide gene for cancer therapy, encode an enzymewhich turns a harmless prodrug to a toxic drug, or that which causesapoptosis and thus should be under tight control until it is needed.

EXAMPLE 16 Reporter Genes

LacZ. This gene is attractive as a reporter gene because it confers theability to identify transduced cells in tissue sections, which arevisualized using X-gal or Bluo-gal (BRL/GIBCO) blue staining withinspecimens isolated over a period of several days or weeks aftertransduction, either from sacrificed animal models or biopsies. LacZanalysis of monolayer cultures and epithelial tissues developed fromprimary keratinocytes grown on a collagen support as raft cultures areboth well established LacZ expression techniques. (S. C. Dollard, et al.Genes and Dev. 1992, 6:1131-1142; & J. L. Wilson et al., Cell GrowthDiff. 1992, 3:471-483). Parker et al., Cell Growth Diff. 1997,8:751-762, Zhao et al., J. Virol. 1997, 71:8832-8840. Similarly, nucleardirected β-galactosids readily detected in rodent airways in vivo afterlipid mediated gene transfer, as well as in murine tracheal explantsfrom animal models using recombinant adenoviral vectors. In addition,LacZ expression is detectable using a quantitative β-galactosidaseactivity assay (Galactolyte; Tropix, Inc.) using a fluorescent substratefor β-galactosidase. The control in such analyses is a plasmid devoid ofHPV sequences, so that it does not replicate, for example pCMV-LacZwhich expresses the LacZ gene from the same promoter employed in theinstant invention. The procedure used for β-galactosidase stainingconsists of washing cells twice with phosphate buffered saline (PBS),fixing the cells in 2% formaldehyde plus 0.2% glutaraldehyde in PBS for10 minutes. The fixed cells are rewashed twice and then stained in 20 mMMgCl₂, 5 mM potassium ferrocyanide, and 40 mg/ml X-gal chromagen(5-bromo-4-chloro-2-indolyl-β-D-galactopyranoside) in PBS. The cells areincubated in staining solution at 37° C. for 2 to 4 hours prior tovisualization.

Luciferase. The luciferase reporter is attractive as a sensitive andquantitative method for transgene expression in lung homogenates frommice, as shown in FIG. 6 for the three plasmid replicon system of theinstant invention. The luciferase gene is cloned into replicons withdemonstrated persistence and therapeutic copy number levels, replacingLacZ. The control in such analyses is preferably pGL3c, an SV40promoter-driven luciferase gene.

Enhanced Green Fluorescence Protein. The EGFP gene is an attractivereporter in cultured cells as well as in vivo due to the ability toidentify transduced cells spectroscopically with quantitive digitallight microscopy. Detection of gene EGFP in airway tissues was reportedby Dr. T. Flotte at the 1997 Cystic Fibrosis Foundation MeetingWilliamsburg, Va., USA. Variants of GFP are now available and cansubstitute readily for one another as reporters in this assay.

EXAMPLE 17 DNA Delivery by Complexing with a Lipid

(2) DNA in complex with GL-67. Lipid 67 (Genzyme Corp., Framingham,Mass.) is an efficient reagent for DNA delivery to mammalian lung andaugments the transgene expression by 100-1,000-fold. Lee et al., HumanGene Therapy 1996, 7:1701-1707. The lipid transfers plasmid-based genesto murine and non-human primate airways at a level roughly equivalent torecombinant adenovirus at an multiplicity of infection (MOI) of 20. S.Cheng, as reported in the Nonviral Vectors Workshop, The North AmericanCF Meeting, Orlando, Fla. 1996). For lipid/DNA instillation into mice, atotal of 650 μg of plasmid is complexed with 108 μg of GL-67 in a finalvolume of 550 μl. The complex is allowed to form without stirring at 30°C. for 5 min prior to instillation into a mouse (100 μl/mouse). For“naked” DNA instillation, the same procedures will be followed in theabsence of GL-67. In previous studies, it has been shown thatadministration of genes by this technique is equivalent in mouse lungswhether the material is instilled by a tracheal cut-down andintracatheter instillation, or simply instilled into the mouse nostrilso that the animal “sniffs” the material into the lungs. Accordingly,one instills 100 μl aliquots of GL-67/plasmid replicon formulations togroups of six mice. Mice are sacrificed at time points (t=2 d, 4 d, 7 d,14 d, 21 d or longer); lungs are snap-frozen in liquid N₂ and pulverizedwith mortar and pestle or Braun tissue homogenizer to a fine powder. Thepowder is processed for luciferase assay. This same approachdemonstrates luciferase signals, even with low levelluciferase-expressing plasmids in the lung (for example, an SV40-drivenplasmid containing luciferase, pGL3c). For measurements of repliconpersistence or replication, lung tissues removed at the same time pointsdescribed above are subjected to an alkaline Hirt extraction protocolsuch as in FIG. 5. After RNase treatment, the low molecular weight DNAis Southern blotted or PCR amplified with appropriate primer pairs afterappropriate restriction enzyme digestion together with Dpn I asdescribed in Example 7. PCR-positive results following Dpn I digestionindicate persistent input DNA and plasmid replication in the mouseairways.

EXAMPLE 18 Determination of Optimum Levels of HPV Replication in vivofor a Preselected Transgene

A successful therapeutic transduction requires that the host immunesystem does not eliminate those cells in which a replicon has beensuccessfully established and a transgene efficiently expressed. Prior totherapeutic administration, replicon persistence and expression areoptionally contrasted between immune-compromised and immune-competentanimal models. Immune compromised models include severe combinedimmunodeficiency (SCID) mice. Optimal levels of HPV replication proteins(as reflected by replicon copy number in vitro) and the possible effectsof E4 and E5 proteins on plasmid persistence in vivo result. Reportergene expression (LacZ or luciferase), and plasmid persistence areassayed over a period of several days or weeks as per the method ofExample 17 for mice tissue.

If the persistence results in SCID mice are consistently better thanresults in immune-competent mice, according to these assays, then thehost immune system is imposing deleterious restriction on reporterexpression and replicon persistence. For instance, if a replicon withouta reporter only persists in SCID mice, then the levels of E1 and E2proteins are likely too high and replicons with lower copy numbers areevaluated as part of the in vivo development. If a replicon without areporter persists in both immune-competent and immune-comprised mice,then the levels of E1 and E2 proteins are likely not a problem intherapeutic administration. If the same replicon except for a reporterhas more extended persistence in SCID mice relative to immune-competentmice, the target of immune reactions is likely to be the reporter geneproduct, and is tested by using a weaker promoter, such as the URRitself, to express the reporter gene, as per Example 15.

Any publications mentioned in this specification are indicative of thelevels of skill in the art relevant to the instant invention. Thesepublications are incorporated herein by reference.

EXAMPLE 19 Adenovirus VAI and VAII Genes

The VA genes are included in most of the preceding example replicons.The presence of these genes enhances the translation of E1 and E2proteins from messages that contain the 5′ untranslated adenovirustripartite leader. Replicons devoid of these genes have a reducedreplication efficiency in transient replication assays. One maydetermine whether the genes also modulate translation when a host cellpromoter such as that for PCNA or DNA polymerase α is used in theabsence of the 5′ untranslated adenovirus tripartite leader sequences.VA genes are optionally deleted if the transgene is an antisense RNA orribozyme, which are not intended for translation.

Any publications mentioned in this specification are indicative of thelevels of skill in the art relevant to the instant invention. Thesepublications are incorporated herein by reference.

Various modifications of the instant invention in addition to thoseshown and described herein will be apparent to those skilled in the artfrom the above descriptions. Such modifications are also fully intendedto fall within the scope of the appended claims.

What is claimed is:
 1. A replicon for delivery of a transgene forepisomal gene expression in a mammalian host cell comprising: atransgene having an open reading frame or other nucleic acid sequencefor transcription into RNA and under the transcriptional control of afirst surrogate promoter and an expression enhancer sequence whereinsaid first surrogate promoter is selected from a group consisting of:HPV-6, HPV-11, HPV-16, HPV-18 and wherein said expression enhancersequence is selected from a group consisting of: HPV-6 upstreamregulatory region, HPV-11 upstream regulatory region, HPV-16 upstreamregulatory region, HPV-18 upstream regulatory region an endogenous HPVupstream regulatory region; a first gene sequence expressing the HPVviral replication initiator protein E1 under the control of anendogenous HPV upstream regulatory region; and a second gene sequenceexpressing the HPV viral replication origin-binding protein E2 under thecontrol of an endogenous HPV upstream regulatory region, wherein saidtransgene, said first and said second sequences are incorporated withinat least one plasmid and less than three plasmids.
 2. The replicon ofclaim 1 wherein said first surrogate promoter and said expressionenhancer sequence are of the same origin.
 3. The replicon of claim 1wherein said expression enhancer sequence is an HPV-11 upstreamregulatory region.
 4. The replicon of claim 1, wherein said transgene isa cystic fibrosis transmembrane regulator.
 5. The replicon of claim 1wherein said transgene is a reporter gene selected to measure replicontransfection and replication efficiencies.
 6. The replicon of claim 1wherein said transgene is selected from a group consisting of:luciferase, LacZ, EGFP, blue fluorescent protein, and other reportergenes.